Promising approaches for the assembly of the catalytically active, recombinant Desulfomicrobium baculatum hydrogenase with substitutions at the active site

Background Hydrogenases (H2ases) are metalloenzymes capable of the reversible conversion of protons and electrons to molecular hydrogen. Exploiting the unique enzymatic activity of H2ases can lead to advancements in the process of biohydrogen evolution and green energy production. Results Here we created of a functional, optimized operon for rapid and robust production of recombinant [NiFe] Desulfomicrobium baculatum hydrogenase (Dmb H2ase). The conversion of the [NiFeSe] Dmb H2ase to [NiFe] type was performed on genetic level by site-directed mutagenesis. The native dmb operon includes two structural H2ase genes, coding for large and small subunits, and an additional gene, encoding a specific maturase (protease) that is essential for the proper maturation of the enzyme. Dmb, like all H2ases, needs intricate bio-production machinery to incorporate its crucial inorganic ligands and cofactors. Strictly anaerobic, sulfate reducer D. baculatum bacteria are distinct, in terms of their biology, from E. coli. Thus, we introduced a series of alterations within the native dmb genes. As a result, more than 100 elements, further compiled into 32 operon variants, were constructed. The initial requirement for a specific maturase was omitted by the artificial truncation of the large Dmb subunit. The assembly of the produced H2ase subunit variants was investigated both, in vitro and in vivo. This approach resulted in 4 recombinant [NiFe] Dmb enzyme variants, capable of H2 evolution. The aim of this study was to overcome the gene expression, protein biosynthesis, maturation and ligand loading bottlenecks for the easy, fast, and cost-effective delivery of recombinant [NiFe] H2ase, using a commonly available E. coli strains. Conclusion The optimized genetic constructs together with the developed growth and purification procedures appear to be a promising platform for further studies toward fully-active and O2 tolerant, recombinant [NiFeSe] Dmb H2ase, resembling the native Dmb enzyme. It could likely be achieved by selective cysteine to selenocysteine substitution within the active site of the [NiFe] Dmb variant. Supplementary Information The online version contains supplementary material available at 10.1186/s12934-023-02127-w.

Supplementary file 3

Separate cloning of the Dmb operon components
The minimal operon of the NiFeSe Dmb H2ase consists of three genes that encode two hydrogenase subunits and a specific maturase. In the first stage of the project, the genes from the hydrogenase operon were cloned and expressed separately in various vector configurations. For this purpose, the designed inserts were amplified and the recombinant constructs for the expression of single proteins were prepared.
Due to the properties of the native hydrogenase genes, and differences between native and heterologous host, DNA fragments encoding signal sequences for periplasmic transport were incorporated into the designed constructs. Four signal sequences were tested: MalE and DsbA in both shorter and longer versions. These DNA fragments were incorporated by means of PCR either into the vector or into the insert sequences.
To prepare the designed synthetic genes with suitable overhangs and vectors enabling the fusion of the DsbA signal to the recombinant protein, PCR reactions were performed.
The LIC cloning method requires the presence of single-stranded, complementary overhangs in the insert and vector sequence. These overhangs are created by T4 DNA polymerase from specifically designed DNA fragments, introduced into the sequence via PCR.
The PCR results were analyzed controlled on agarose gels. All planned reactions were conducted successfully with a single band of desired product observed.

PCR amplification with addition of signal sequences
Inserts for different vectors were prepared with LH mutants U493C, U493M, U493STOP, as well as SH and HMP as templates. The main goal of this PCR round was to create a range of primary inserts with terminal sequences complementary for 5 vectors used in this study. Vectors pRSF and pMCSG53 have identical flanking sequences for the subcloned genes, thus one version of the insert can be used for annealing with either of them. Vector pMCSG53 carries ampicillin resistance and allows for overproduction of proteins with fused N-terminal His-tags. Vector pRSF carries resistance to ampicillin and allows for the overproduction of non-tagged proteins. The origins of replication of these vectors are compatible. Thus, a pair can be used for coexpression of genes from two plasmids in one bacterial cell. The same principle applies for the pMCSG92 and pMCSG93 vector pair.
Vector 93 carries kanamycin resistance and the resulting recombinant protein has an additional His-tag preceded by a TEV site on the C-terminus. Vector 92 carries ampicillin resistance. The recombinant protein variants obtained with the use of v92 recombinant constructs have C-terminal His-tags preceded by a TEV site. Vector DsbA is based on the vector 93 backbone and requires the addition of the DsbA signal sequence encoding DNA fragment to the insert in order to be annealed.

Annealing and transformation
PCR reactions were cleaned-up from excess dNTPs and subjected to reaction with T4 DNA polymerase. The alignment of the insert/vector pairs, as well as insert modifications obtained by PCR reactions, are listed in Suppl. Tab.1. Annealing and transformation were performed according to the protocol described in the Methods section of the manuscript.
The protocol for bacterial growth and the genes expression is provided in the main manuscript. Transformants were observed after 18 hours of incubation for all reactions (Suppl. Tab.1). A total of 36 types of recombinant E. coli variants were obtained.

Expression
Three colonies were selected from each transformation well and used to inoculate 1 ml of LB. Bacterial cultures were spun down after 18 hours from IPTG induction of the recombinant gene expression. Bacterial cells from each well were lysed, 4 µl samples were taken and analyzed by SDS-PAGE.
Constructs with positive results from the expression screening were chosen for plasmid DNA isolation and DNA sequencing. Based on the sequencing results, plasmid DNAs isolated from positive bacterial clones were used as templates for the next round of PCR, aiming for the construction of the designed operons.
Out of the obtained recombinant bacterial clones (with confirmed biosynthesis of POIs), twenty-nine recombinant constructs were purified and sequenced. All the constructs are listed in Suppl. Tab.1, along with gel numbers and lanes for reference (Suppl. Fig.1A and B). 4 Suppl. Tab.1. Properties of the three main target protein variants, produced in the first stage of the research and marked positive after DNA sequencing. Location of His-tag and TEV site are marked as N-term (located at N-terminus) or C-term (located at C-terminus). The POI biosynthesis level is evaluated using a threepoint scale (1-low, 2-medium, 3-high). The biosynthesis of the obtained recombinant protein variants was investigated by SDS-PAGE. Gel and lane numbers corresponding to the investigated recombinant constructs are provided below in Suppl. Fig.1  The main purpose of this stage was to produce inserts for assembling operons with RBS 514 or RBS 517 sequences. Thirty six PCR reactions were prepared with the previously confirmed constructs as DNA templates. PCR products were analyzed using agarose gel electrophoresis. Two PCR reactionsdesigned for SECIS element reconstructionwere negative.

Annealing and transformation
Thirty four specific PCR products were purified from an excess of dNTPs and subjected to incubation with T4 DNA polymerase. Operons consisting of two genes, with RBS 514 or 517, in various vectors, were assembled (LH-SH, LH-HMP, and SH-HMP) to result in twenty transformation reactions (Suppl. Tab.2).
Both kanamycin-and ampicillin-resistant colonies were observed after overnight incubation.

Expression
Two bacterial colonies were picked from each agar well and grown on a small scale. After

IMAC purification
Based on the results of the POI production screening, twenty five recombinant bacterial clones were chosen for IMAC purification experiments (Suppl. Tab.3). Most of the selected constructs code for two His-tagged proteins and so show two bands when analyzed by SDS-PAGE. Thus, two bands were detected in the analyzed samples and show rather the occurrence of both proteins than pairing of the subunits (Suppl. Fig.3

Production of hydrogenase complex via co-expression of three-piece operon elements
Native Dmb is derived from a strictly anaerobic, Gram-negative bacterium.
Hydrogenases, per se, are vulnerable to oxidizing species. Thus, it is legitimate to apply anaerobic growth conditions for hydrogenase biosynthesis. In fact, such an approach was previously used by others to express heterologous hydrogenases genes from organisms Suppl. Tab.4. Constructs aligned for co-expression. Constructs marked in red manifested a slow growth rate and two projects with HMP_v92 proved to be toxic upon induction (cell lysis).

Expression and IMAC analysis
After lysis, total protein samples were taken and IMAC purification was performed.
Samples from cultures grown in aerobic (O2) and anaerobic (O2) conditions were analyzed side by side (Suppl. Tab.5), (Suppl. Fig.4). Constructs with LH U493C and HMP appeared to be especially toxic upon expression. No significant changes in gene expression and protein production were observed under anaerobic conditions. . C493-HMP constructs appeared to be especially toxic. No significant changes in expression and protein production were observed under anaerobic conditions.

Production of hydrogenase complex via in vitro assembly
Due to the high toxicity of certain protein constructs, or very low growth rate of some

PCR amplification
Twenty five PCR reactions were prepared. The obtained PCR products were analyzed using agarose gel electrophoresis.

Annealing and transformation
Specific PCR products were purified from the excess of dNTPs and subjected to reaction

Expression analysis
Two colonies were picked from each agar well (if possible) and grown in 1 ml of LB. After . It was hypothesized that producing the LH subunit without the endoproteolytically cleavable C-terminal extension could simplify complex maturation and subunit assembly (Fig.31).
Suppl. Fig.7. Layouts of a two-piece operon with truncated hydrogenase (top) and three-piece operon (bottom). Bacteria after transformation were plated on agar plates with corresponding antibiotics.

Annealing and transformation
Co-transformant alignments are presented in Suppl. Tab.8.
Suppl. Tab.8. Alignment of constructs selected for co-expression in aerobic (O2) and anaerobic (O2) conditions. Gel and lane numbers from corresponding SDS-PAGE analyses are noted. All DNA constructs presented in the

Expression analysis
Two colonies were picked from each agar well (if possible) and grown in 1 ml of LB. After 18 hours from IPTG induction, cells were spun down and lysed. Samples with a volume of 4 µl were taken after lysis. The collected samples were analyzed by SDS-PAGE (Suppl.  Transformation of E. coli with SH_v53 and SH_vDsbA constructs was performed. Transformants were grown in 3 ml of LB medium with a range of supplements at 37˚C. After 17 hours from induction with IPTG, 1.5 ml of each culture was processed and the rest was frozen for further experiments. SDS-PAGE was performed for the analysis of protein samples after IMAC purification (Suppl. Fig.9).

gel XXI
Suppl. Fig.9. Optimization of growth conditions for the improvement of the SH subunit solubility. Protein samples obtained after IMAC were analyzed by SDS-PAGE. Abbreviations: sup -M9 "Pink" Medium Metal Supplement, Fe -iron supplementation, PLP -pyridoxal phosphate supplementation, cys -cysteine supplementation. Small subunit (SH) abbreviation is placed corresponding to the predicted POIs migration in gel.

Small subunit solubilization and in vitro Fe-S clusters incorporation
Since the observed levels of expressed SH are high, but only a small amount can be

SH HMP
Suppl. Tab.10. Constructs chosen for the biosynthesis of the POI in anaerobic conditions. Amount of POI in the soluble fraction and IMAC elution was controlled via SDS-PAGE (Suppl. Fig.11 Fig.11. SDS-PAGE analysis of the soluble POIs obtained after expressed of various DNA constructs. Large subunit (LH), small subunit (SH) and hydrogenase maturation protease (HMP) abbreviations are placed corresponding to the predicted POIs migration in gel.

Production of hydrogenase complex via expression of two-piece operon elements
Due to the truncated version of LH, (C or M)493 were tested positive for assembly with separately expressed SH (SH_v53) constructs. The operons containing both elements were constructed.

PCR amplification
Twelve PCR reactions were prepared with the previously obtained and verified constructs as DNA templates. The products of PCR reactions were analyzed by gel electrophoresis.

Annealing and transformation
PCR amplification products were purified from the excess of dNTPs and subjected to incubation with T4 DNA polymerase. DNA fragments were annealed and used for E. coli transformation, resulting in 5 transformation attempts (Suppl. Tab.11). Ampicillin-resistant clones were observed after overnight incubation. rbs517_LH M493_499STOP_His-tag XXV 6

Expression
Bacterial colonies (Suppl. Tab.11) were picked from each agar plate wells and grown in 1 ml of LB. After reaching the desired A600, the recombinant gene expression was induced upon the addition of IPTG. After 18 hours from the induction, cells were spun down and lysed. Samples were analyzed using SDS-PAGE (Suppl. Fig.12).

gel XXV
Suppl. Fig.12. SDS-PAGE analysis of the POIs biosynthesis levels obtained after expression of the selected DNA constructs, described in Suppl. Tab. 11. Large subunit (LH), small subunit (SH) and hydrogenase maturation protease (HMP) abbreviations are placed corresponding to the predicted POIs migration in gel.

Purification
For the activity testing, protein samples (Suppl. Tab.12) were purified by IMAC and then subjected to SEC chromatography to exclude aggregates. Fractions corresponding to the soluble, multimeric form of the enzyme were combined. Samples were analyzed using SDS-PAGE (Suppl. Fig.13).